Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure, and for convenience the references are listed in the list of references appended hereto.
Over the past decade, there have been significant advances in the development of selective biosensors based on the use of DNA as a biorecognition element. While the majority of DNA based sensors are designed to detect complementary DNA, many recent reports have demonstrated that single-stranded DNA can also form intricate tertiary structures that allow it to selectively bind to non-DNA targets (so called aptamers)1,2 or perform catalysis of chemical reactions.3,4 To date, over 100 DNA sequences have been reported for facilitating many types of chemical transformations.5 In spite of having very limited chemical functionalities, deoxyribozymes that perform catalysis with surprising efficiency have been reported in a number of studies.6 For example, a small DNA enzyme known as 10-23 performs site-specific RNA cleavage with a very impressive kcat of ˜10 min−1.7 It is clear that the lack of a 2_-hydroxyl group in DNA relative to RNA is not a detriment to catalytic performance. Furthermore, the catalytic capabilities of DNA can be enhanced through the use of metal ions8 and small-molecule cofactors9 as well as through modification with chemical functionalities that are useful for catalysis.10 Furthermore, when compared to ribozymes, deoxyribozymes are easier to prepare and more resistant to chemical and enzymatic degradation, and therefore, properly engineered and catalytically efficient DNA enzymes are very desirable elements for the construction of rugged biosensors.
Allosteric ribozymes and deoxyribozymes have tremendous potential for wide-ranging applications in the diagnostic, biosensing and drug screening fields. The use of deoxyribozymes with fast catalytic rates and large turnover numbers allows for the engineering of effective allosteric DNA enzymes for practical applications where rapid enzymatic action is essential. To engineer catalytic DNA probes for detection directed applications, it is very desirable to use DNA enzymes that can couple enzymatic activity with fluorescence signaling capability so that easy and fast detection can be performed in real time without the need for time-consuming separation steps.